PROJECT SUMMARY/ABSTRACT Ozone (O3) continues to be of great public health concern with more than 1/3 of the U.S. population, 122 million people, currently living in areas exceeding the National Ambient Air Quality Standard (NAAQS), which are exposure levels known to cause inflammatory responses in humans. O3 is highly reactive and known to oxidize biomolecules, including unsaturated lipids, such as cholesterol. Yet, how O3-induced chemical reactions translate into intracellular effects presents a knowledge gap. O3-derived products of cholesterol include electrophiles, such as oxysterol, which have the ability to form adducts with nucleophilic centers of proteins, thus affecting cellular signaling. The overall objective of this application is to determine how formation of oxysterols and oxysterol-protein adducts link O3-induced chemical reactions with biological effects. We developed experimental protocols in which airway epithelial cells (ECs) are treated with alkyne-modified O3- derived oxysterols followed by reacting the cell lysates with an azido biotin reagent under ?click? cycloaddition conditions, resulting in the biotinylation of any protein that forms a covalent bond with alkyne-modified oxysterol. This biotinylated protein mixture can be ?pulled down? for proteomic analysis of the ?adductome? or individual oxysterol-protein adduct formation. Using a proteomic screen of oxysterol-protein adducts formed in ECs, we identified NLRP2 as a potential target. Specific Aim 1 will expand these initial studies, characterize the overall protein ?adductome? generated by O3-derived oxysterol in ECs, and focus on the role of oxysterol- adducted NLRP2 in O3-induced pro-inflammatory responses. Specific Aim 2 will focus on how O3-derived oxysterols affect macrophage function and whether similar to ECs, oxysterols form protein adducts in macrophages, thus affecting cellular function. Using a co-culture system composed of ECs and macrophages, this aim will also determine whether oxysterols formed at or near EC membranes communicate with macrophages. Specific Aim 3 will focus on the relationship between 7-dehydrocholesterol (7-DHC), the last step during cholesterol biosynthesis, and O3-induced inflammation. 7-DHC is more susceptible to O3-induced oxysterol formation and we have evidence that increased lung 7-DHC levels correlate with O3-induced inflammation in humans in vivo. Furthermore, we show that modifying 7-DHC levels by commonly prescribed small molecule antidepressants enhance O3-induced inflammation. Using linked in vitro, mouse in vivo, and human in vivo experiments this aim is designed to determine how pharmacologically modulating pulmonary 7- DHC levels could increase the susceptibility to O3-induced inflammation. The findings developed in this study will uncover novel interactions between oxidized lipids and modification of cellular function in the context of O3 exposure and foster a better understanding of how commonly prescribed drugs could sensitize to O3-induced inflammation.